Our research focuses on bacterial infections in muco-obstructive lung diseases, a collection of genetic and acquired illnesses including cystic fibrosis (CF), non-CF bronchiectasis, primary ciliary dyskinesia (PCD), and chronic obstructive pulmonary disease (COPD). Individuals with muco-obstructive lung diseases experience progressive respiratory function decline due to recurrent and chronic bacterial infections. Pseudomonas aeruginosa (Pa) is an opportunistic bacterial pathogen and the leading cause of recalcitrant chronic infection in individuals with muco-obstructive lung disease. Our lab seeks to cultivate a more thorough understanding of chronic Pa lung infection pathogenesis using state-of-the-art model systems, including in vitro, in vivo, and ex vivo approaches. Current projects focus on host-microbial and polymicrobial interaction in the context of the lung environment, the impact of antibiotic usage on microbial pathogenesis and population dynamics, and changes in host innate immune metabolism and signaling that contribute to disease progression.
Antibiotic treatment failure is widespread with Pseudomonas infections in patients with muco-obstructive lung disease. Antibiotic tolerance is a poorly understood phenomenon that significantly contributes to persistent infections in patients with muco-obstructive lung diseases. Our long-term goal is to determine how Pa genotype/phenotype and host environment factors such as hyper-concentrated mucus contribute to antibiotic tolerance. Furthermore, we are currently investigating the phenotypic changes that occur within the population following antibiotic treatment using ex vivo clinical specimens and in vitro models.
Evolution of Pseudomonas during muco-obstructive lung disease
The evolution of Pa populations in the lung contributes to antibiotic treatment failure, persistence, and respiratory decline. We aim to identify factors in the host environment that contribute to the evolution and genotypic/phenotypic diversification of Pa.
Loss of quorum sensing (QS) is a common phenotype observed in Pa during chronic infection. QS regulates an extensive network of genes in Pa important for pathogenesis, such as virulence factor production and biofilm formation. Moreover, QS in Pa is required for the establishment of infection in model systems. Nevertheless, loss of QS is correlated with chronic infection and declining lung function in CF. We aim to investigate how the loss of QS provides a fitness advantage and contributes to worsening disease.
Coordinated Regulation of Pseudomonas Virulence
The ability of Pa to cause this wide variety of diseases depends on the expression of an array of virulence factors. We have discovered that synthesis of the second-messenger signaling molecule adenosine 3′, 5’-cyclic monophosphate (cyclic AMP or cAMP) is required for Pa virulence. Specifically, cAMP generates a transcriptional response by binding to and activating the transcription factor Vfr (Virulence factor regulator). We have demonstrated that cAMP is specifically required for Vfr DNA binding activity and that vfr expression is autoregulated and cAMP-dependent. Transcriptional analyses indicate that the cAMP-Vfr complex controls the expression of nearly 200 genes, the majority of which encode acute or invasive virulence factors required for host colonization, surface motility, and the secretion and delivery of exotoxins. Based on these results, we propose that the cAMP-signaling pathway plays a central role in detecting the host environment and initiating a coordinated pathogenic response.
Work in the lab has focused on understanding the three main mechanisms that control intracellular levels of cAMP in bacteria: synthesis, degradation, and transport. We are specifically interested in understanding how each of these processes are regulated and the role they play in controlling Pa virulence gene expression in vivo.
In addition to studying the mechanisms that control intracellular cAMP levels, we have discovered that expression of vfr is subject to inhibition by the alginate extracellular polysaccharide regulatory pathway. The production of an alginate capsule is an important Pa adaptation that occurs during (and potentially facilitates) the chronic phase of infection in muco-obstructive lung disease. While the molecular mechanism of vfr inhibition remains to be determined, our discovery provides direct evidence for the inverse regulation of acute and chronic virulence factors during chronic infection. Recent work in the lab implicates a post-transcriptional control mechanism mediated by two homologous RNA binding proteins, RsmA and RsmF. Ongoing research is aimed at understanding the molecular basis of post-transcriptional regulation in Pseudomonas.
Cell Culture Model of Muco-obstructive Lung Disease Infection
Primary human bronchial epithelial (HBE) cultures, grown at the air-liquid interface, are a well-established and dynamic model for studying the human airway mucosa. We have exploited this system to generate a biologically relevant mucus infection model. We are currently using a multi-omics approach to investigate host epithelial and bacterial changes that occur during infection of the mucus layer. Preliminary findings indicate that key aspects of human airway infection are recapitulated. In addition, we are utilizing this model system to study the impact of antimicrobial and host-directed therapeutics on infection and disease-related outcomes.
Murine Model of Chronic Lung Infection
The generation of a robust and reproducible chronic pulmonary bacterial infection model in mice is a critical requirement for studying pathogenesis and therapy in muco-obstructive lung diseases. Collaboratively, we are working to develop a murine model of chronic bacterial infection. As part of this effort, we are systematically evaluating both bacterial and host phenotypic and genotypic characteristics that enable chronic Pseudomonas aeruginosa and Staphylococcus aureus lung infection. Using these murine model systems, we hope to better understand the impact of in vivo bacterial adaptation and host immune modulation during chronic infection. In addition, these studies will advance our understanding of the complex host-microbial interactions that drive patient outcomes and shape drug efficacy in muco-obstructive lung diseases.
Microbiome of Chronic Muco-Obstructive Lung Disease
Defective innate airway defense mechanisms, by definition, render individuals susceptible to colonization and infection by a spectrum of bacterial and viral pathogens. The presence of these organisms can precipitate further deterioration of existing defenses through continuous assault or punctuated events (acute exacerbations), resulting in a vicious cycle that drives disease progression.
To better understand the complex infection biology of chronic airway diseases, we use next-generation sequencing methods to detect, identify, and quantify bacterial and viral organisms in airway-derived specimens. Using this metagenomic approach, we have characterized the CF lung microbiome as a function of age, disease severity, exacerbation, and antibiotic therapy. Our studies have revealed that the earliest detectable infections in infants and young children involve anaerobic bacteria likely derived from oral aspiration. Subsequent infection by opportunistic pathogens, including Staphylococcus aureus and Pa, result in lung microbial communities that are highly refractive to antibiotic treatment. These observational clinical studies have created new areas of research in the lab aimed at understanding the role of polymicrobial interactions and community succession in muco-obstructive lung diseases. Ongoing collaborative studies include a detailed examination of the oral-lung microbiome axis in CF and the role of the nasal microbiome in acute exacerbation frequency in COPD.
Cystic Fibrosis Related Diabetes (CFRD)
CF-related diabetes (CFRD) is a common comorbidity affecting 20% of adolescents and 50% of adults with CF. CFRD results in accelerated rates of pulmonary decline and earlier mortality compared to CF with normal glycemic control. Individuals with CFRD are more prone to pulmonary infections that are associated with earlier mortality. The bacterial pathogens Pseudomonas aeruginosa and Staphylococcus aureus are primarily responsible for pulmonary infection in CFRD. Whereas P. aeruginosa typically becomes the dominant pathogen later in life, individuals with CFRD experience a resurgence of S. aureus. Importantly, CFRD infections are associated with increased antibiotic failure rates. The driving factors behind the re-emergence of S. aureus are currently unknown. Understanding the effects of CFRD on infection dynamics and disease pathogenesis and progression will inform more effective therapeutic strategies.
In response to the SARS-CoV-2 pandemic, our lab, in partnership with laboratories in the UNC Adams School of Dentistry and the UNC School of Medicine, launched the DELTA Translational Core to process, store and distribute COVID-19 clinical samples in support of research efforts across the globe. In addition to providing support for other studies, Dr. Wolfgang leads a multidisciplinary research team studying innate immune metabolism in the lungs of severely ill COVID-19 patients using a systems biology-based approach. This deep phenotyping effort has demonstrated that severe COVID-19 has a complex pathophysiology with multiple etiologies that require distinct therapeutic approaches. Current efforts are aimed at identifying targets for host-directed disease therapies and molecular and clinical biomarkers to differentiate disease trajectories.